There is now strong data supporting our early hypothesis that the reduced incidence of enteric diseases in breastfeeding infants is due, in part, to protection by human milk glycans. Glycans, including glycoproteins, glycolipids, mucins, and glycosaminoglycans, contain complex oligosaccharide structures attached to proteins, lipids, and other molecular backbones. These complex carbohydrate moieties are synthesized by the many glycosyltransferases in the mammary gland; those glycans with homology to cell surface glycoconjugate pathogen receptors may inhibit pathogen binding, thereby protecting the nursing infant. Rotavirus and HIV infections in infants are introduced primarily through oral inoculation, and we discovered that these pathogenic viruses are strongly inhibited by specific high molecular weight human milk glycans in vitro. A subset of the human milk glycosaminoglycans, a chondroitin sulfate, and a component of the human milk sulfated glycosphingolipid fraction each strongly inhibit HIV in solid phase assays and in human leukocytes. A human milk glycoprotein, lactadherin (46 kDa), strongly inhibits rotavirus infection of MA 104 cells, and this molecule accounts for all of the inhibitory activity of human milk against rotaviruses in vitro. This proposal focuses on characterizing these actively antiviral glycans, using state-of-the-art instrumental analytical techniques that have recently become available and can provide detailed understanding of the molecular structure of complex glycan moieties. The molecular mechanisms will be studied to include the inhibition of multiple strains of the pathogens by the whole molecules. This will be followed by research to identify the smallest active moieties of the active molecules, and confirming their mechanism of pathogen inhibition. These studies are designed to culminate in a plan to synthesize the active moieties through genetic engineering of Kluyveromyces lactis, yeast occurring naturally in many dairy products of the human diet, and which has proved amenable to synthesis of other active human milk glycans. The availability of synthetic human milk glycans suitable for oral consumption that inhibit HIV or rotavirus could have large impact on these important threats to public health.